Blastomere movement correlates with ploidy and mosaicism in early-stage human embryos after in vitro fertilization

Zygote ◽  
2021 ◽  
pp. 1-15
Author(s):  
Xiaodong Zhang ◽  
Jingwei Yang ◽  
Wei Han ◽  
Chong Li ◽  
Guoning Huang
Keyword(s):  
Laser Scanning ◽  
Early Stage ◽  
Screening Method ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Human Embryos ◽  
Cell Stage ◽  
Preimplantation Genetic Testing ◽  
Vitro Fertilization

Summary Embryos undergo chaotic division and decrease in quality on day 3 with a reduction in the rates of subsequent blastocyst formation. Disordered cleavage causes a deterioration in embryonic quality, here we assessed the relationship between an cleavage model in first mitosis and the chromosomal status of human embryos, and discuss the potential biological and clinical implications for the cleavage model as a single parameter that can be used to assess embryonic quality. Thirty-two infertile couples, with normal karyotypes and who underwent their first IVF cycle were recruited to donate one normal two-cell-stage embryo each for this study between 2019 and 2020. Twenty-eight two-cell embryos underwent preimplantation genetic testing of each blastomere, and four chaotic-division embryos were stained with Hoechst and cultured in a confocal laser-scanning microscopy incubator system. This system showed high specificity and PPV but low sensitivity and NPV using the CM in the prediction of euploidy, indicating that CM could be considered a screening method for embryo selection; additional observational studies using the CM to select transferable embryos are needed before it can be used in clinical practice.

scholarly journals Increased mitochondrial distribution in early-cleaving embryos indicate successful pre-implantation development

2018 ◽  
Vol 14 (4) ◽  
pp. 512-514
Author(s):  
Nor Shahida Abdul Rahman ◽  
Mimi Sophia Sarbandi ◽  
Wan Hafizah Wan Jusof ◽  
Zolkapli Eshak ◽  
Salina Othman ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Close Association ◽  
Mitochondrial Fission ◽  
Polar Body ◽  
Blastocyst Stage ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Cell Stage ◽  
Second Polar Body

The timing of the first zygotic cleavage is an accurate predictor of embryo quality. Embryos that cleaved early have higher developmental viability compared to their late counterparts. During embryonic development, cleavage is affected by cellular metabolic processes performed by mitochondria and its synergistic interaction with endoplasmic reticulum (ER). However, in depth study on differences of mitochondria and ER ultrastructures in early- cleaving (EC) versus late- cleaving (LC) embryos is limited. This study compares mitochondria and ER ultrastructures of EC versus LC embryos using Confocal Laser Scanning Microscopy (CLSM) and Transmission Electron Microscopy (TEM). Embryos were obtained from female ICR superovulated mice, 28-30 hours post hCG. Two-cell embryos were categorized as early-cleaving (EC), while zygotes with the second polar body and two pronuclei present were categorized as late-cleaving (LC). The LC embryos were cultured in vitro until the 2- cell stage. In EC embryos, mitochondria were mostly found at the perinuclear region and closely associated with dense ER. Meanwhile, mitochondria of LC embryos were distributed uniformly within the cytoplasm. Mitochondrial fluorescence intensity was significantly higher in EC versus LC [(18.7 ± 0.4) versus (14.6 ± 0.4)] x 105 pixel, (p<0.01). Development to the blastocyst stage was also significantly higher in EC compared to LC embryos (96.7% versus 60.9%) (p<0.01). Higher viability of EC embryos is attributed to the close association of their mitochondria to ER. This contributed to better mitochondrial fission, resulting in enhanced energy generating processes and preimplantation development. 

scholarly journals 286SPERM-OOCYTE INTERACTION DURING IN VITRO FERTILIZATION IN THE HORSE

2004 ◽  
Vol 16 (2) ◽  
pp. 263
Author(s):  
J.L. Tremoleda ◽  
T.A.E. Stout ◽  
B.M. Gadella ◽  
B. Colenbrander
Keyword(s):  
In Vitro Fertilization ◽  
Laser Scanning ◽  
Transvaginal Ultrasound ◽  
Polar Body ◽  
Molecular Probes ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Vitro Fertilization

In vitro fertilization (IVF) has proven to be a surprisingly unsuccessful way of producing horse embryos. The aim of this study was to investigate the interaction between sperm and the cumulus oocyte complex (COC) during IVF. In experiment 1, three IVF conditions were tested: (A) COCs recovered from slaughtered mares were categorized with respect to cumulus morphology (C: compact, n=86, or E: expanded, n=55) and matured in TCM199 containing 0.01IU/mL porcine FSH and equine LH (IVM); after IVM, the oocytes were denuded and those with a visible polar body were incubated with sperm (IVF) in the presence or absence of 150ng/mL progesterone (P4) to induce the acrosome reaction (AR); (B) IVM oocytes from C-COCS were denuded (n=52) or not (n=67) before IVF in the presence of P4;; (C) in vivo-matured oocytes (n=15) recovered by transvaginal ultrasound-guided aspiration from preovulatory follicles 32h after the donor mare was treated with hCG, were fertilized in vitro in the presence of P4. In all cases, IVF was performed with frozen-thawed, Percoll-selected sperm from a single stallion, at a final concentration of 1×106spermatozoa/ml in fertil-TALP for 20h (Parrish et al., 1988 Biol. Reprod. 38, 1171–1180). In experiment 2, the possibility that semen cryopreservation or stallion critically influenced IVF was examined by incubating denuded IVM oocytes with fresh or frozen/thawed sperm from the same (fresh;; n=17 for both C- and E-COCs and frozen-thawed; n=12 and 21 for C and E-COCs, respectively) or one other stallion (Fresh;; n=12 and 19 and frozen-thawed; n=12 and 19 for C and E-COCs, respectively), in the presence of P4 for 20h. In both experiments, the resulting sperm-oocyte complexes were fixed, permeabilized and labelled with fluorescein-conjugated peanut agglutinin (EY Laboratores, San Mateo, CA, USA) and ethidium homodimer (Molecular Probes, Eugene, OR, USA) to stain the acrosomal membrane and DNA, respectively, so that membrane status and position of the sperm within the oocyte investments could be detected by confocal laser scanning microscopy. The total number of sperm bound per oocyte was compared between treatments using one-way ANOVA with pair-wise multiple comparison (Bonferroni t-test). Despite binding to the zona pellucida (ZP), neither fresh nor frozen/thawed sperm from either stallion acrosome-reacted or penetrated any oocytes, irrespective of cumulus morphology at the onset of IVM, denudation prior to IVF or the presence of P4. However, more sperm bound to the ZP of cumulus-denuded IVM oocytes (65±32 and 62±28 [mean±sd] for C and E-COCs, respectively), than cumulus-intact IVM (5±4) or in vivo-matured oocytes (23±17: P&lt;0.001). None of the other factors investigated affected bound sperm numbers. In all cases, ZP-bound sperm failed to AR in the classical fashion, and all oocytes remained arrested at the MII stage. In summary, fertilization failed because sperm did not acrosome-react after binding to the ZP. It is concluded that failure to adequately activate stallion sperm is an important obstacle to successful IVF in horses.

scholarly journals Methods to Evaluate Skin Penetration In Vitro

2019 ◽  
Vol 87 (3) ◽  
pp. 19 ◽  
Author(s):  
Zsikó ◽  
Csányi ◽  
Kovács ◽  
Budai-Szűcs ◽  
Gácsi ◽  
...  
Keyword(s):  
Drug Development ◽  
Laser Scanning ◽  
Early Stage ◽  
Pharmaceutical Research ◽  
Skin Penetration ◽  
Experimental Models ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Tape Stripping

Dermal and transdermal drug therapy is increasing in importance nowadays in drug development. To completely utilize the potential of this administration route, it is necessary to optimize the drug release and skin penetration measurements. This review covers the most well-known and up-to-date methods for evaluating the cutaneous penetration of drugs in vitro as a supporting tool for pharmaceutical research scientists in the early stage of drug development. The aim of this article is to present various experimental models used in dermal/transdermal research and summarize the novel knowledge about the main in vitro methods available to study skin penetration. These techniques are: Diffusion cell, skin-PAMPA, tape stripping, two-photon microscopy, confocal laser scanning microscopy, and confocal Raman microscopic method.

scholarly journals A New gcrR-DeficientStreptococcus mutansMutant for Replacement Therapy of Dental Caries

2013 ◽  
Vol 2013 ◽  
pp. 1-7 ◽  
Author(s):  
Wenting Pan ◽  
Tiantian Mao ◽  
Qing-an Xu ◽  
Jin Shao ◽  
Chang Liu ◽  
...  
Keyword(s):  
Replacement Therapy ◽  
Laser Scanning ◽  
Early Stage ◽  
Morphological Characteristics ◽  
Laser Scanning Microscopy ◽  
Negative Regulator ◽  
Confocal Laser ◽  
Adhesion Assay

Background.gcrRgene acts as a negative regulator related to sucrose-dependent adherence inS. mutans. It is constructive to test the potential capacity of mutans withgcrRgene deficient in bacteria replacement therapy.Methods. In this study, we constructed the mutant by homologous recombination. The morphological characteristics of biofilms were analyzed by confocal laser scanning microscopy.S. mutansUA159 and the mutant MS-gcrR-def were inoculated, respectively, or together for competitive testing in vitro and in rat model.Results. Adhesion assay showed that the adhesion ability of the mutant increased relative to the wild type, especially in the early stage. MS-gcrR-def out-competedS. mutansUA159 in vitro biofilm, and correspondingly coinfection displayed significantly fewer caries in vivo. The former possessed both a lower level of acid production and a stronger colonization potential thanS. mutansUA159.Conclusion. These findings demonstrate that MS-gcrR-def appears to be a good candidate for replacement therapy.

Vacuoles Induced in Mammalian Cells by Helicobacter Cytotoxin are Acidic Organelles

1990 ◽  
Vol 48 (3) ◽  
pp. 168-169
Author(s):  
M. H. Chestnut ◽  
C. E. Catrenich
Keyword(s):  
Acridine Orange ◽  
Mammalian Cells ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Ulcer Disease ◽  
Gastroduodenal Disease ◽  
H Pylori

Helicobacter pylori is a non-invasive, Gram-negative spiral bacterium first identified in 1983, and subsequently implicated in the pathogenesis of gastroduodenal disease including gastritis and peptic ulcer disease. Cytotoxic activity, manifested by intracytoplasmic vacuolation of mammalian cells in vitro, was identified in 55% of H. pylori strains examined. The vacuoles increase in number and size during extended incubation, resulting in vacuolar and cellular degeneration after 24 h to 48 h. Vacuolation of gastric epithelial cells is also observed in vivo during infection by H. pylori. A high molecular weight, heat labile protein is believed to be responsible for vacuolation and to significantly contribute to the development of gastroduodenal disease in humans. The mechanism by which the cytotoxin exerts its effect is unknown, as is the intracellular origin of the vacuolar membrane and contents. Acridine orange is a membrane-permeant weak base that initially accumulates in low-pH compartments. We have used acridine orange accumulation in conjunction with confocal laser scanning microscopy of toxin-treated cells to begin probing the nature and origin of these vacuoles.

scholarly journals Effect of dissolution rate and subsequent ion release on cytocompatibility properties of borophosphate glasses

2019 ◽  
Vol 5 (1) ◽  
pp. 85-97
Author(s):  
Nusrat Sharmin ◽  
Mohammad S. Hasan ◽  
Md. Towhidul Islam ◽  
Chengheng Pang ◽  
Fu Gu ◽  
...  
Keyword(s):  
Dissolution Rate ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Ion Release ◽  
Scanning Calorimetry ◽  
Cell Culture Study ◽  
Borophosphate Glasses ◽  
Mg63 Cells

AbstractPresent work explores the relationship between the composition, dissolution rate, ion release and cytocompatibility of a series of borophosphate glasses. While, the base glass was selected to be 40mol%P2O5-16mol%CaO-24mol%MgO-20mol%Na2O, three B2O3 modified glass compositions were formulated by replacing Na2O with 1, 5 and 10 mol% B2O3. Ion release study was conducted using inductively coupled plasma atomic emission spectroscopy (ICP-AES). The thermal scans of the glasses as determined by differential scanning calorimetry (DSC) revealed an increment in the thermal properties with increasing B2O3 content in the glasses. On the other hand, the dissolution rate of the glasses decreased with increasing B2O3 content. To identify the effect of boron ion release on the cytocompatibility properties of the glasses, MG63 cells were cultured on the surface of the glass discs. The in vitro cell culture study suggested that glasses with 5 mol% B2O3 (P40B5) showed better cell proliferation and metabolic activity as compares to the glasses with 10 mol% (P40B10) or with no B2O3 (P40B0). The confocal laser scanning microscopy (CLSM) images of live/dead stained MG63 cells attached to the surface of the glasses also revealed that the number of dead cells attached to P40B5 glasses were significantly lower than both P40B0 and P40B10 glasses.

scholarly journals Cephalosporin nitric oxide-donor prodrug DEA-C3D disperses biofilms formed by clinical cystic fibrosis isolates of Pseudomonas aeruginosa

2019 ◽  
Vol 75 (1) ◽  
pp. 117-125 ◽  
Author(s):  
Odel Soren ◽  
Ardeshir Rineh ◽  
Diogo G Silva ◽  
Yuming Cai ◽  
Robert P Howlin ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Clinical Isolates ◽  
Nitric Oxide Donor ◽  
Confocal Laser ◽  
Broth Microdilution Method

Abstract Objectives The cephalosporin nitric oxide (NO)-donor prodrug DEA-C3D (‘DiEthylAmin-Cephalosporin-3′-Diazeniumdiolate’) has been shown to initiate the dispersal of biofilms formed by the Pseudomonas aeruginosa laboratory strain PAO1. In this study, we investigated whether DEA-C3D disperses biofilms formed by clinical cystic fibrosis (CF) isolates of P. aeruginosa and its effect in combination with two antipseudomonal antibiotics, tobramycin and colistin, in vitro. Methods β-Lactamase-triggered release of NO from DEA-C3D was confirmed using a gas-phase chemiluminescence detector. MICs for P. aeruginosa clinical isolates were determined using the broth microdilution method. A crystal violet staining technique and confocal laser scanning microscopy were used to evaluate the effects of DEA-C3D on P. aeruginosa biofilms alone and in combination with tobramycin and colistin. Results DEA-C3D was confirmed to selectively release NO in response to contact with bacterial β-lactamase. Despite lacking direct, cephalosporin/β-lactam-based antibacterial activity, DEA-C3D was able to disperse biofilms formed by three P. aeruginosa clinical isolates. Confocal microscopy revealed that DEA-C3D in combination with tobramycin produces similar reductions in biofilm to DEA-C3D alone, whereas the combination with colistin causes near complete eradication of P. aeruginosa biofilms in vitro. Conclusions DEA-C3D is effective in dispersing biofilms formed by multiple clinical isolates of P. aeruginosa and could hold promise as a new adjunctive therapy to patients with CF.

scholarly journals Use of electromagnetic stimulation on an Enterococcus faecalis biofilm on root canal treated teeth in vitro

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Beatriz H. D. Panariello ◽  
Justin K. Kindler ◽  
Kenneth J. Spolnik ◽  
Ygal Ehrlich ◽  
George J. Eckert ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Root Canal ◽  
Laser Scanning ◽  
Confocal Imaging ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Microscopy Imaging ◽  
Electromagnetic Stimulation ◽  
Root Canal Disinfection

AbstractRoot canal disinfection is of utmost importance in the success of the treatment, thus, a novel method for achieving root canal disinfection by electromagnetic waves, creating a synergistic reaction via electric and thermal energy, was created. To study electromagnetic stimulation (EMS) for the disinfection of root canal in vitro, single rooted teeth were instrumented with a 45.05 Wave One Gold reciprocating file. Specimens were sterilized and inoculated with Enterococcus faecalis ATCC 29,212, which grew for 15 days to form an established biofilm. Samples were treated with 6% sodium hypochlorite (NaOCl), 1.5% NaOCl 1.5% NaOCl with EMS, 0.9% saline with EMS or 0.9% saline. After treatments, the colony forming units (CFU) was determined. Data was analyzed by Wilcoxon Rank Sums Test (α = 0.05). One sample per group was scored and split for confocal laser scanning microscopy imaging. There was a significant effect with the use of NaOCl with or without EMS versus 0.9% saline with or without EMS (p = 0.012 and 0.003, respectively). CFUs were lower when using 0.9% saline with EMS versus 0.9% saline alone (p = 0.002). Confocal imaging confirmed CFU findings. EMS with saline has an antibiofilm effect against E. faecalis and can potentially be applied for endodontic disinfection.

scholarly journals Cocktail of carbohydrases from Aspergillus niger: an economical and eco-friendly option for biofilm clearance from biopolymer surfaces

AMB Express ◽  
2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Arashdeep Kaur ◽  
Sanjeev Kumar Soni ◽  
Shania Vij ◽  
Praveen Rishi
Keyword(s):  
Aspergillus Niger ◽  
Laser Scanning ◽  
Statistical Modelling ◽  
Laser Scanning Microscopy ◽  
Enzyme Treatment ◽  
Confocal Laser ◽  
Abiotic Surfaces ◽  
Enzyme Cocktail ◽  
Natural Variant

AbstractBiofilm formation on both biotic and abiotic surfaces accounts for a major factor in spread of antimicrobial resistance. Due to their ubiquitous nature, biofilms are of great concern for environment as well as human health. In the present study, an integrated process for the co-production of a cocktail of carbohydrases from a natural variant of Aspergillus niger was designed. The enzyme cocktail was found to have a noteworthy potential to eradicate/disperse the biofilms of selected pathogens. For application of enzymes as an antibiofilm agent, the enzyme productivities were enhanced by statistical modelling using response surface methodology (RSM). The antibiofilm potential of the enzyme cocktail was studied in terms of (i) in vitro cell dispersal assay (ii) release of reducing sugars from the biofilm polysaccharides (iii) the effect of enzyme treatment on biofilm cells and architecture by confocal laser scanning microscopy (CLSM). Potential of the enzyme cocktail to disrupt/disperse the biofilm of selected pathogens from biopolymer surfaces was also assessed by field emission scanning electron microscopy (FESEM) analysis. Further, their usage in conjunction with antibiotics was assessed and it was inferred from the results that the use of enzyme cocktail augmented the efficacy of the antibiotics. The study thus provides promising insights into the prospect of using multiple carbohydrases for management of heterogeneous biofilms formed in natural and clinical settings.

scholarly journals pH and Reduction Dual-Responsive Bi-Drugs Conjugated Dextran Assemblies for Combination Chemotherapy and In Vitro Evaluation

Polymers ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 1515
Author(s):  
Xiukun Xue ◽  
Yanjuan Wu ◽  
Xiao Xu ◽  
Ben Xu ◽  
Zhaowei Chen ◽  
...  
Keyword(s):  
Combination Chemotherapy ◽  
Laser Scanning ◽  
Rational Design ◽  
A549 Cells ◽  
Chemotherapeutic Agents ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Tumor Resistance ◽  
Oxidized Dextran

Polymeric prodrugs, synthesized by conjugating chemotherapeutic agents to functional polymers, have been extensively investigated and employed for safer and more efficacious cancer therapy. By rational design, a pH and reduction dual-sensitive dextran-di-drugs conjugate (oDex-g-Pt+DOX) was synthesized by the covalent conjugation of Pt (IV) prodrug and doxorubicin (DOX) to an oxidized dextran (oDex). Pt (IV) prodrug and DOX were linked by the versatile efficient esterification reactions and Schiff base reaction, respectively. oDex-g-Pt+DOX could self-assemble into nanoparticles with an average diameter at around 180 nm. The acidic and reductive (GSH) environment induced degradation and drug release behavior of the resulting nanoparticles (oDex-g-Pt+DOX NPs) were systematically investigated by optical experiment, DLS analysis, TEM measurement, and in vitro drugs release experiment. Effective cellular uptake of the oDex-g-Pt+DOX NPs was identified by the human cervical carcinoma HeLa cells via confocal laser scanning microscopy. Furthermore, oDex-g-Pt+DOX NPs displayed a comparable antiproliferative activity than the simple combination of free cisplatin and DOX (Cis+DOX) as the extension of time. More importantly, oDex-g-Pt+DOX NPs exhibited remarkable reversal ability of tumor resistance compared to the cisplatin in cisplatin-resistant lung carcinoma A549 cells. Take advantage of the acidic and reductive microenvironment of tumors, this smart polymer-dual-drugs conjugate could serve as a promising and effective nanomedicine for combination chemotherapy.

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